Experiment 14:  Paternity analysis using PCR

Literature

• Chuah, S.Y., Tan, W.F., Yap, K.H., Tai, H.E., & S.T Chow (1994). Analysis of the D1S80 locus by amplified fragment length polymorphism technique in the Chinese, Malays, and Indians in Singapore. Forens. Sci. Int. 68, 169-180.
• Kasai, K., Nakamura, Y., & R. White (1989). Amplification of a variable number of tandem repeats (VNTR) locus (pMCT118) by the polymerase chain reaction (PCR) and its application to forensic science. J. Forensic Sci. 35, 1196-1200.
• Kloosterman, A.D., Budowle, B. & P. Daselaar (1993). PCR-amplification and detection of the human D1S80 VNTR locus. Int. J. Leg. Med. 105, 257-264.
• Mullis, K.B. (1990). Ein Nachtfahrt und die Polymerase-Kettenreaktion. Spektrum der Wissenschaft. 6, 60-67.
• Nakamura, Y., Carlson, M., Krapcho, K., & R. White (1988). Isolation and mapping of a polymorphic DNA sequence (pMCT118) on chromosome 1p [D1S80]. Nucl. Acids Res. 16, 9364.
• Narayana, S. (1991). Applications of restriction fragment length polymorphism. Annals Clin. Lab. Sci. 21, 291-296.
• Saiki, R.K. et al. (1985). Enzymatic amplification of beta-globin sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 230, 1350-1354.

Objective

     To amplify the locus D1S80 with PCR and electrophoretocally separate the PCR product to determine whether or not the man is the child's father.

Theory: PCR

     PCR is a technique that can be used to amplify a section of DNA. This was developed in the mid-80s by Kary Mullis (Saiki et al., 1984; Mullis, 1990) and has revolutionized DNA sequencing. PCR is based on a simple principle and exploits the features of DNA replication. Two important points of PCR are a) that it can target a specific range of DNA and b) its multiplicity. The starting material of a PCR is DNA that contains the section to be amplified. ssDNA is acquired by heat-denaturing the dsDNA and used as a template.

     Also required are DNA polymerase and 2 short primers specific to a short section on both strands of your sequence. Under the right reaction conditions and in the presence of the 4 dNTPs, the DNA polymerase will lengthen a complementary chain along the denatured ss template, forming 2 new DNA strands containing the target sequence. Each strand contains a new binding site for the primers, which can anneal after the next cycle of heat-denaturation, allowing synthesis to start over again. The end result of PCR after n cycles is a reaction mixture containing a theoretical maximum of 2ⁿ (really 2ⁿ - 2n) dsDNA molecules containing the DNA range between the primers plus the primer binding site. The temperature cycle is automated and illustrated in the figure above.

   PCR entails the following 3 steps:

     i.  Denaturation of dsDNA: 95蚓.
     ii.  Annealing of the primer: 55-65蚓
     iii. DNA synthesis using a heat-stable DNA polymerase: ~72蚓

   The first 3 rounds of amplification are shown in the figure to the left.

Theory: DNA polymorphism in the human genome

     A DNA polymorphism is the occurrence of two or more alleles containing the same gene in a given population, usually with a frequency >1%. It is generally identified using restriction enzymes, producing a so-called restriction fragment length polymorphism (RFLP) (Narayanan, 1991), which happens when fragments of different lengths are produced by digestion with restriction enzymes. (See figure 3.) This is caused by a VNTR - variable number of tandem repeats - see figure 4.

Figure 3. (R)  RFLP. In a), we see a Southern blot of 3 samples of genomic DNA that have been cleaved with MspI. The different patterns have been numbered 1-3. In b), we see an illustration of the corresponding chromosomes (A and B). The hybridization sequence of the probe has been marked.  M is the MspI restriction site. Figure 4. (L)  VNTR polymorphism. a) shows a Southern blot with the genomic DNA samples from figure 3, this time cut with HinfI and HaeIII. b) shows the corresponding chromosomes A-F. H1 is the HinfI restriction site; H3 is that for HaeIII. The black box is for the homologous region of the probe. (?)

     VNTR loci are good targets for PCR, since the primers (loci) flanking the target sequences are known. (?) Since the number of repeats results in different fragment lengths, the PCR product can be analyzed via electrophoresis to positively identify the corresponding allele - see figure 5.

Figure 5.   VNTR analysis with PCR. The VNTR regions were
amplified with PCR using primers flanking the polymorphous region.
The PCR product is then electrophoretically separated by size.

In this experiment, the locus D1S80 (Nakamura et al., 1988) will be analyzed with PCR to determine paternity (Kasai et al., 1988; Kloosterman et al., 1993). This locus contains a VNTR make up of hexadecamer (16-membered) repeats. The size of the repeats coupled with the static distribution of the allele lengths in the population (16-40x repetition) makes this locus quite suitable for individual identification (Kloosterman et al., 1993).

Procedure

PCR reaction
     For the following components (Kloosterman et al., 1993), use a sterile PCR Eppendorf container and pipette the (small 無) volumes directly into the DNA solution (ie, not onto the wall).

Centrifuge shortly and place in the Thermocycler heat block. Use the following program for the PCR:
   2 min 95蚓
   30 x (60s 66蚓  |  90s 72蚓  |  60s 95蚓)
   5 min 72蚓
   cool to 30蚓

Finish by stopping with 3 無 blue stop solution. Before applying the samples to the PAG, heat for 5 minutes at 95蚓 to denature them, then shock-cool them so they can't reanneal properly and store on ice.